According to WHO, safe blood is a universal right, which means that blood that will not cause any harm to the recipient and that has been fully screened and is not contaminated by any blood-borne disease such as HIV, hepatitis, malaria, or syphilis. Hepatitis B is one of the most common diseases transmitted by blood and individuals with chronic infection have a high risk of developing liver cirrhosis and hepatocellular carcinoma. The current study looks at the sero-prevalence of Hepatitis B infection amongst healthy blood donors coming to Blood Bank, J N Medical College Hospital, AMU, Aligarh during the period 2006-2010. All blood donations (voluntary or replacement) collected over this period were included. Collected samples were screened for hepatitis B surface antigen (HBsAg; Hepalisa, J. Mitra) and Enzyme linked immunosorbent assay (ELISA), to determine the seropositivity of infection in the donors. Of the 53422 units of blood collected over a 5- year period, 24040 (45%) were from voluntary and 29382 (55%) from replacement donors. Among the donors, 1636(3.1%) were positive for Hepatitis B, of which 1546(94.5%) were males and 90 (5.5%) were female donors. The seroprevalence of HBsAg was relatively high (3.1%) in our study when compared to the reported rates in other parts of country and only 5.5% females were positive for HBsAg as compared to 94.5% of males. Safety of the blood supply is dependent on collecting blood from voluntary donors from low-risk populations, screening donated blood for transmissible infections and avoiding unnecessary transfusions. These activities need to be carried out by a well-coordinated blood transfusion service with quality control being implemented at all levels and efforts should be made to increase the number of voluntary donors and reduce replacement donations to a minimum.